The long-term aim of this program is to elucidate the chemistry and physiology of the acyl CoA dehydrogenases and the electron transfer flavoprotein. Work in progress involves mechanistic studies, electron spin resonance and electrochemical studies and structural investigations on the two flavoproteins employing substrate analogs and covalently modified derivatives of the two proteins. The interaction between the dehydrogenase and the ETF is being investigated using stable derivatives of the dehydrogenase with modified carboxyl groups. In these derivatives the reductive half-reaction is unaffected but the dehydrogenase does not react with ETF. Photochemically generated and substrate-generated EFT semiquinone has been characterized and the environment of the radical is currently being investigated by EPR spectroscopy using the technique of matrix ENDOR. The reaction of the substrate-reduced and dithionite-reduced dehydrogenase with the EFT is being studied in order to elucidate the mechanism of the reaction. The reductive half-reaction of the dehydrogenase is being investigated further using pseudosubstrates. The redox potentials of ETF flavin have been determined. Potentiometric studies of the dehydrogenase and dehydrogenase-dead-end inhibitor complexes are in progress.